Sugar is a key source of energy for multicellular organisms, and its efflux across the membrane is critical to many physiological processes, including blood glucose maintenance and milk production. SWEET transporters (SWEETs) are novel membrane proteins that mediate sugar export. SWEETs are also prototypes of the large MtN3 membrane protein clan, which includes notable members such as mitochondrial pyruvate carrier, PQ-loop transporters and the KDEL receptor. Eukaryotic SWEETs, together with their half transporter bacterial homolog, SemiSWEET, are a unique model system to study the widely observed duplication-fusion in membrane protein evolution. Despite the importance of SWEETs in sugar utilization and MtN3 proteins in mitochondrial function, lysosomal amino acid homeostasis, and ER protein retention, we do not understand SWEET and MtN3 mechanisms at the molecular level. To overcome a major barrier to progress-the lack of a structural framework to guide our mechanistic understanding of transport-we have solved high-resolution structures of two SemiSWEET proteins, in two distinct conformational states: outward open and occluded. We will leverage this structural data to gain a detailed understanding of the structure and function of SWEETs and, more broadly, of MtN3s. Specific Aim 1: Elucidate the structural basis of sugar transport by SemiSWEET using X-ray crystallography and biophysical methods. These studies will help us understand the physical basis of sugar transport by SemiSWEETs. Specific Aim 2: Determine the first crystal structure of a eukaryotic SWEET. This structural information will provide a blueprint for the transport process and help elucidate the evolution of membrane transport proteins with internal symmetry. Specific Aim 3: Dissect the transport mechanism of SWEETs and MtN3s through functional studies. Fundamental aspects of SWEET-mediated transport will be elucidated and then extended to members of the MtN3 clan. OVERALL IMPACT: The proposed research will reveal the structural basis of sugar transport by SWEETs, elucidate their transport mechanism, and accelerate exploration of MtN3s as therapeutic targets to treat diabetes, cancer and lysosomal storage diseases.